Modulation of lncRNA H19 enhances resveratrol‐inhibited cancer cell proliferation and migration by regulating endoplasmic reticulum stress

Abstract The phytoalexin resveratrol exhibits anti‐tumour activity in many types of cancer. In this study, we showed that resveratrol suppressed the survival of gastric tumour cells both in vivo and in vitro. Resveratrol promoted apoptosis, autophagy and endoplasmic reticulum (ER) stress in a dose‐dependent manner. RNA‐seq analysis showed that multiple cell death signalling pathways were activated after resveratrol treatment, while the use of ER stress activators (tunicamycin and thapsigargin) in combinatorial with resveratrol led to further inhibition of cancer cell survival. Results also showed that resveratrol altered the expression of several long non‐coding RNAs (lncRNAs), including MEG3, PTTG3P, GAS5, BISPR, MALAT1 and H19. Knockdown of H19 in resveratrol‐treated cells further enhanced the effects of resveratrol on apoptosis, ER stress and cell cycle S‐phase arrest. Furthermore, the migratory ability of resveratrol‐treated cells was dramatically decreased after H19 knockdown. In conclusion, resveratrol inhibited cancer cell survival, while knockdown of lncRNA H19 resulted in increased sensitivity to resveratrol therapy.


siRNA Transfection
To knockdown lncRNA H19 in SGC7901 cells, siRNA 5′-CCCACAACAUGAAAGAAACTT -3′ and 5′-GCUAGAGGAACCAGACCUUTT -3′were used in this study. As a control for these studies, a non-silencing negative siRNA sequence obtained from Genecreate (Wuhan, China) was used. The siRNA was transfected into cells using Lipofectamine 3000 reagent (Invitrogen, Shanghai, China) according to the manufacture's guidelines. A final concentration of the siRNA was 100 nM. After being incubated with lncRNA H19 siRNA for 24 hours, cells were re-transfected for another 48 hours.
Resveratrol was added to the cells when lncRNA H19 knockdown for 48 hours and treated SGC7901 cells for another 24 hours. Finally, the knockdown cells were harvested after 72 hours.

Drugs
Resveratrol was purchased from Sigma-Aldrich. The resveratrol powder was dissolved in DMSO into 50 mM stock solution and stored at -20℃ condition, then diluted in culture media prior to experimentation. The complete medium DMEM with the same volume of DMSO was used as the control.

Cell Cycle and Apoptosis Assay
Cell cycle distribution and apoptosis were examined via a flow cytometer (Fortessa, BD Biosciences, New York, USA), and the results were analyzed by ModFit software. 6×10 5 cells were seeded in the 6 cm dishes and harvest after 24 h with various concentration resveratrol treatment or 72 h with lncRNA H19 siRNA transfection.
For cell cycle analysis, cell pellets were washed with cold PBS and fixed in 70% ice-cold ethanol in -20℃ overnight. The fixed cells were stained in PI/RNase Staining Buffer Solution (BD Biosciences, New York, USA) for 30 min at room temperature in the dark according to the manufacturer's instruction.
An Annexin V/PI double staining apoptosis detection kit was used to detect cell apoptosis. After resveratrol and/or lncRNA H19 knockdown treatment, the adherent and suspended cells were harvest and washed with cold PBS. Then cells were incubated in 100 µL 1×binding buffer containing Annexin V-FITC and PI at room temperature for 15 min in the dark according to the manufacturer's instruction.
Thereafter, 400 µL 1×binding buffer was added and the flow cytometer as mentioned above was utilized to detect the apoptosis.

Quantitative Real-Time PCR
qRT-PCR was used to detect the mRNA expression in treated SGC7901 cells. In brief, the TRIzol reagent (Tiangen Biotech, Beijing, China) was used to isolate the total RNA and the All-in-One cDNA synthesis SuperMix (Bimake, Shanghai, China) was used to reverse-transcribed 2 μg total RNA into cDNA according to the manufacturer's protocol. Then, the 2×SYBR Green qPCR Master Mix (Bimake, Shanghai, China) was used to detect the mRNA expression following the manufacturer's protocol on a CFX96 real-time PCR detection system and GAPDH was used as a reference gene. The relative expression of the target genes was calculated using the 2 -ΔΔCt method.

Figure S3
GSEA analysis was performed on RNA-seq data. p53-related pathways were upregulated in resveratrol-treated cells. Myc-associated pathways were downregulated in resveratrol-treated cells.

Figure S4
Expression of lncRNAs in resveratrol (Res)-and H19 siRNA-treated SGC7901 cancer cells. SGC7901 cells were treated with H19 siRNA for 72 h and 50 μM resveratrol for 24 h, with gene expression determined by qRT-PCR. Reported values are mean ± SEM. *p < 0.05, **p <0.01 and ***p <0.001 indicate significant differences compared with the control group.